PROJECT SUMMARY Normal function of the lower urinary tract (LUT) requires coordination of neuronal activity to allow bladder filling for urine storage followed later by bladder contraction to accomplish urine expulsion at an appropriate time and place. Deficiencies in the development of the neural elements that mediate bladder control can lead to problems in patients such as neurogenic bladder, incontinence, urinary retention, or bladder pain. These LUT diseases result in reduced quality of life for patients, increase healthcare costs, and burden the health care system. While we know that the sacral elements of the peripheral nervous system, which participate in regulation of these bladder processes, include dorsal root ganglion (DRG) sensory neurons and autonomic (motor) pelvic neurons, there are many gaps in our knowledge regarding how these neurons develop and how deficits during development lead to congenital defects in bladder control. By surveying gene expression during development of pelvic ganglia in mice we have identified up-regulation of serotonin receptors that are also expressed in sensory neurons of developing DRG. Mice with loss of function mutations in these serotonin receptors, specifically the serotonin type 3 receptor (5-HT3), have abnormal development of nerves in the bladder wall and later exhibit urinary retention. Our preliminary work in isolated cultures of sacral neural crest stem cells indicates that perturbations of 5-HT3 signaling disrupt neuronal differentiation. We postulate that the bladder deficiencies observed in 5-HT3 mutant mice occur not only as the result of hyper-arborization of developing nerve terminals that lack 5-HT3 signaling, but also as a consequence of altered cell fate specification leading to imbalances among types of developing DRG and pelvic ganglia neurons. Cell fate specification is a novel function that has not previously been ascribed to 5-HT3 and has potential for pronounced impact on the types of neurons formed during development of bladder innervation. Such alterations in neuronal differentiation would predispose to deficiencies in bladder control and add to the potential for increased susceptibility to bladder pain. Three aims are proposed that will elaborate roles for 5-HT3 signaling during development of DRG and pelvic ganglia neurons that innervate the bladder. In Aim 1 we will use genetic and pharmacologic approaches to determine when 5- HT3 signaling is required to develop and maintain normal bladder innervation. In Aim 2 we will use Cre:LoxP lineage tracing studies in Htr3a gain and loss of function mutants to define the developmental processes and time periods that are affected by 5-HT3 signaling. In Aim 3 we will determine whether disruptions of 5-HT3 signaling during development increase vulnerabilty to bladder inflammatory pain. These studies will generate mechanistic knowledge of deficits in neural development that lead to bladder disease and will aid urologists in sculpting personalized therapies for patients.